Skin is the outer covering of mammals which protects all the deeper layers of the body. As such, it is equipped with a powerful immune system to ward off challenges from the external environment. Complement is a vital component of that immune protection. However, it is a two edged sword because aberrant activity can also damage host tissue. This is particularly true of the membrane attack complex (MAC) which can cause bystander lysis of host cells. Therefore agents that can appropriately inhibit aberrant complement activation will have important therapeutic benefits in skin disorders where self damage occurs.
Complement is activated through two major pathways, the classical and the alternative. For a summary of the complement pathways, and the stages inhibited by the invention described here, see Drawing 1.
Bernstein et al. in U.S. Pat. No. 4,007,270 claimed that aurin tricarboxylic acid was a complement inhibitor. However no chemical analysis of the claimed product was carried out. Subsequent analytical studies based on the synthetic method described in the patent showed that the products consisted mostly of a mixture of high molecular weight materials of uncertain structure (Gonzalez et al. 1978; Kushman and Kanamathareddy 1990; Lee et al. 2012a; PCT/IB2012/053608; U.S. patent application Ser. No. 13/195,216). Moreover, these high molecular weight components have powerful side effects which would render them unsuitable for human administration, including inhibition of protein nucleic acid interactions (Gonzales et al. 1979).
It has previously been demonstrated that aurin tricarboxylic acid itself (ATA), and the closely related derivatives aurin quadracarboxylic acid (AQA), and aurin hexacarboxylic acid (AHA), block classical pathway formation at the stage of C9 binding to C5b678 so that formation of the MAC (C5b-9) cannot take place (Lee at al. 2012a). It has further been demonstrated that ATA binds to Factor D, the protease which cleaves membrane bound Factor B. This creates a block in the alternative pathway subsequent to the stage where properdin (Factor) P has bound C3b and Factor B to form the complex PC3bB. Cleavage of Factor B by Factor D to form PC3bBb (C3 convertase) is required for the pathway to proceed. Therefore ATA also blocks the alternative pathway at the C3 convertase stage (Lee et al. 2012b).
The invention described here is novel in that it utilizes low molecular weight components of the aurin tricarboxylic acid complex (ATAC) to block complement activation in the skin. These include aurin tricarboxylic acid itself (ATA, MW 422), aurin quadracarboxylic acid (AQA, MW 573) and aurin hexacarboxylic acid (AHA, MW 857). These active molecules can be isolated from the mixture obtained after synthesis by the method of Cushman and Kanamathareddy (1990) by passing the crude synthetic product through a 1 kDa filter. They occur in the approximate proportion of 78% AHA, 15% AQA, and 7% AHA.
ATA itself, or the complex ATAC, by inhibiting complement activation, can be expected to treat successfully many skin diseases where aberrant complement activation is reported to occur (Kotnick 2010). Although the causes of such activation may be diverse, the fundamental reactions are common. They involve activation of local phagocytes, release of cytotoxins and inflammatory mediators, and formation of the autolytic MAC. Therefore, ATA and its derivatives will be successful in treating these diverse conditions by inhibiting unwanted complement activation at the C3 convertase and C9 addition to C5b-8 stages.
Pemphigus is one example. In this potentially fatal disorder, there is an autoimmune attack against desmoglien, the adhesive protein which forms the attachment of adjacent epidermal cells. The classical and alternative pathways are both activated with consequent formation of the membrane attack complex (Kawana et al. 1989), indicating that ATA and its derivatives will be effective therapeutic agents.
Dermatitus herpetiformis is a condition which is characterized by an extremely itchy rash. It is linked to gluten intolerance and immune attack against the protein epidermal transglutaminase (Preisz et al. 2005). The lesions are characterized by depositions of IgA accompanied by C3, Factor P and Factor B, indicating activation of the alternative pathway of complement (Seah et al. 1973). By inhibiting the alternative pathway at both the C3 convertase and C9 addition stages, ATA and its derivatives will be effective therapeutic agents.
Psoriasis is a common skin condition which is characterized by an immune response. There is a proliferation of keratinocytes which release inflammatory cytokines such as TNFα, as well as the complement proteins C3, Factor B, C7, and C9 (Ballantini et al. 2011). Since TNFα increases C9 synthesis, it contributes to formation of the membrane attack complex (Kotnik 2010). Specifically, it has been shown that there is deficiency in psoriatic arthritic patients of membrane bound CD59, the protective agent against self attack by the MAC (Triolo et al. 2003). Therefore ATA and its derivatives will be effective therapeutic agents.
Discoid lupus erythematosis is an autoimmune disorder which is highly exacerbated by sunlight. It is currently treated with topical steroids, indicative of the effectiveness of immune blockade. By blocking aberrant complement activation, ATA and its derivatives will be effective therapeutic agents.
Irritant or allergic dermatitis occurs when the immune system of the skin attacks an allergen or other irritant. Keratinocytes can become damaged by this excessive attack. Aberrant complement activation will be ameliorated by treatment with ATA and its derivatives.
Primary cicatricial alopecia (PCA) is a skin disorder in which epithelial hair follicle stem cells are damaged or destroyed by inflammatory events (Harries et al. 2009). The affected stem cells reside in the outer root sheath of hair follicle bulges (Harries et al. 2009). This is an area hypothesized to be immunologically privileged (Meyer et al. 2008). Loss of such immunological privilege results in immune attack, so that hair follicles are replaced by scar tissue, with loss of hair (Harries et al. 2010). A mainstay of treatment is topical steroids. Since inflammation activates the complement system. By blocking this aberrant complement activation, ATA and its derivatives will be effective therapeutic agents.
Androgenic alopecia (baldness) is the most common form of hair loss in males. It begins after puberty, with initial losses occurring in the temporal and occipital areas. It typically advances to baldness covering the entire scalp except for a rim extending around the peripheral regions. The cause is generally acknowledged to be a vulnerability of hair follicles in these areas to androgens, particularly dihydrotestosterone (DHT) (Garza et al. 2012). DHT interacts with the androgen receptor (AR) and it is high levels of this receptor that are presumed to create the vulnerability. The gene for AR is located at Xq11-Xq12. Genetic analyses have shown that variants in the receptor are associated with androgenic alopecia (Hillmer et al. 2005) and especially with a nearby gene named ectodysplasin receptor 2A (EDA2R) (Prodi et al. 2008). The product of the gene is a transmembrane protein of the tumor necrosis factor receptor superfamily. To date there is no explanation as to why activation of these receptors by DHT should result in disappearance of hair follicles in androgenic alopecia. Treatments for baldness have been developed based on reducing the substrate for these receptors, or increasing scalp circulation. Examples include minoxidil, an arterial dilator that has been used systemically and topically to promote hair growth (U.S. Pat. No. 5,030,442). Finasteride, which blocks 5-alpha reductase conversion of testosterone to DHT, has been approved for hair loss. However, none of these approaches, or others that are totally empirical, provide any insight as to the mechanism that actually causes disappearance of hair follicles. Nevertheless, we have demonstrated in this invention that ATA and its derivatives not only arrest hair loss, they stimulate the reappearance of hair follicles with new hair growth in areas that have become bald. Application of ATA at a concentration of 1-5 micrograms per ml 2-3 times daily, results in an abundance of new hair follicles appearing in previously bald areas after 4-6 weeks of treatment. This indicates that baldness can to some degree reflect hair follicle suppression rather than hair follicle destruction. One possible explanation is that ATA and its derivatives are blocking complement attack of an enduring nature, thus permitting keratinocyte follicular re-growth.
Seborrheic dermatitis and dandruff are caused by an excessive loss of corneocytes from the outer layer of the epidermis. Immune dysfunction is suspected (Mills et al. 2012). The corneocytes adhere to each other creating flakes which are then shed. The pathogenesis appears to result from interactions between scalp skin, cutaneous microflora and the cutaneous immune system (Kerr et al. 2011). A proposed cause is Malassezia fungi (Gemmer et al. 2002). Increased levels of inflammatory markers are detected in biopsy specimens including IL-1α and IL-1RA (Kerr et al. 2011). By blocking the consequent complement activation, ATA and its derivatives will be effective therapeutic agents.
Alopecia areata is a condition in which there is hair loss, usually from the scalp. It is characterized by a lymphocytic infiltration around vulnerable follicles, so that the hair growth disappears. However there may be remissions so that hair may grow again. The standard treatment is topical corticosteroids appropriate to an inflammatory response, or minodoxil, a capillary dilator which stimulates hair growth. By blocking the complement activation which accompanies inflammation, ATA and its derivatives will be effective therapeutic agents.